Purification and characterization of five different α subunits of guanine‐nucleotide‐binding proteins in bovine brain membranes

We have purified five different α subunits of guanine‐nucleotide‐binding proteins (G proteins) from bovine brain membranes as active forms bound to guanosine 5′‐[γ‐thio]triphosphate (GTP[γS]). All the purified α subunits were interacted with βγ subunits and served as a substrate for pertussin‐catalyzed ADP‐ribosylation. Based on the findings of immunoblot analyses using specific antibodies raised against various α subunits of G proteins, three of them were identified as α i‐1 .α i‐2 and α i‐3 3, and the other two were classified into α 0 type. One of the α 0 ‐type proteins was the most abundant in the brain membranes (termed α 0 ), and the other (α 0 2) appeared to differ from α 0 in its proteolytic digestion data. The physiological properties of these purified GTP[γS]‐bound α subunits towards adenylate cyclase and atrial muscarinic K + channels were studied. The nucleotide‐bound forms of α i‐1 , α i‐2 . α i‐3 and α 0 2 inhibited the adenylate cyclase activity of S49 cyc − membranes which had been reconstituted with GTP[γS]‐treated G s ; this inhibition appeared to be mainly competitive with the activated G s , α i‐1 having the most potent inhibitory activity among them. GTP[γS]‐bound α 0 , however, could not inhibit the G s ‐stimulated activity at all. On the other hand, all the GTP[γS]‐bound α subunits activated atrial muscarinic K + channels, accompanied by a lag time, at picomolar concentrations. The βγ subunits resolved from G proteins also activated the K + channels without a lag time at nanomolar concentration. The maximum activation by the βγ subunits appeared to be more potent than that by any of the a subunits. These results suggest that α and βγ subunits might activate the K+ channels by mechanisms different from each other.